Biomarker for determining mitochondrial damage in friedreich&#39;s ataxia

ABSTRACT

Compositions and methods for screening for a disease or a disorder associated with a deficiency in frataxin in a subject using biomarkers for diseases or disorders associated with a deficiency in frataxin are disclosed. The compositions and methods include determining the acetylation status of mito-chondrial proteins. Also disclosed are methods of detecting progression of a disease or a disorder associated with a deficiency in frataxin in a subject and methods of monitoring effectiveness of a therapy for diseases or disorders associated with a deficiency in frataxin.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Publication no. 2015/0132769,filed on Sep. 2, 2014, which claims priority to InternationalPublication no. PCT/US2013/028609, filed on Mar. 1, 2013, which claimspriority to U.S. provisional application No. 61/605,783, filed on Mar.2, 2012 and U.S. provisional application No. 61/607,918, filed on Mar.7, 2012, all of which are incorporated herein by reference in theirentireties.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under HL085098 awardedby the National Institutes of Health. The government has certain rightsin the invention.

BACKGROUND OF THE DISCLOSURE

The present disclosure relates generally to compositions and methods forscreening for diseases or disorders associated with a deficiency infrataxin. More particularly, the present disclosure relates to methodsof screening for a disease or disorder associated with a deficiency infrataxin, as well as methods of screening for therapeutic agents for usein treating a disease or disorder associated with a deficiency infrataxin, or damage from reactive oxygen species to mitochondrialproteins affecting the respiratory chain, or increased mitochondrialprotein acetylation affecting mitochondrial function, as well as methodof screening for therapeutic agents for use in treating a disease ordisorder associated with a deficiency in frataxin.

Friedreich's Ataxia (FRDA) is an autosomal recessive mitochondrialdisorder caused by a homozygous triplet nucleotide repeat (GAATTC)expansion in intron 1 of the FXN gene located on chromosome 9q21.11.This intronic expansion causes impaired transcription of the FXN geneand, consequently, a pathological deficiency of the FXN gene product,frataxin. Frataxin is targeted to the mitochondrial matrix, where it isknown to act as an iron-binding protein and participates in the properassembly and function of iron-sulfur cluster (ISC) dependent proteinsincluding complexes I, II, and III of the respiratory chain andaconitase of the tricarboxylic acid (TCA) cycle. Thus, frataxindeficiency severely compromises both cellular respiration and overallmitochondrial function leading to energetic stress and ATP deficiency.Although patients develop multisystem disease including earlyspinocerebellar degeneration, ataxia, and diabetes, the primary cause ofdeath is heart failure for nearly 85% of those afflicted. Similarly,although the phenotypes of the neuron-specific enolase (NSE) and musclecreatine kinase (MCK) Cre conditional mouse models of FRDA differ, bothmodels develop a fatal cardiomyopathy and impaired activity ofiron-sulfur cluster-dependent respiratory complexes consistent with thehuman disease.

Recent work has demonstrated that lysine acetylation is a highlyconserved and abundant post-translational modification withinmitochondria that is responsive to nutrient availability and maycontribute to the physiological adaptations of reduced caloric intake.Multiple investigations have demonstrated a role for reversiblemitochondrial enzyme deacetylation and, specifically, the NAD⁺-dependentdeacetylase SIRT3, in the regulation of fatty acid oxidation, the TCAcycle, electron transport via respiratory complexes I and II, andoverall oxidative metabolism. SIRT3-mediated deacetylation has recentlyemerged as a major mechanism regulating the activity of mitochondrialoxidative and intermediary metabolism. SIRT3 is also uniquely poised torespond to the flux of mitochondrial NAD⁺ and NADH, which is determined,in large part, by the capacity of the respiratory chain to oxidize NADH.This capacity is severely decreased in FRDA, as well as in othermitochondrial defects such as cytochrome c oxidase (complex IV)deficiency, causing an accumulation of NADH and, consequently, a redoxstate of perceived nutrient excess.

Defects in cellular respiration may be inherited as mitochondrialdisease, or acquired over a lifetime via somatic mutations, and arelinked to many conditions including neurodegenerative disease, diabetes,heart failure, cancer and in the aging process in general. Theinvolvement of cellular respiration in numerous common human pathologiesemphasizes the need for greater understanding of the pathophysiologicalprocesses that occur in response to respiratory chain compromise.Accordingly, there exists a need to develop biomarkers of diseases ordisorders associated with a deficiency in frataxin.

SUMMARY OF THE DISCLOSURE

The present disclosure relates generally to methods and kits forscreening for diseases and disorders associated with a deficiency infrataxin. More particularly, it has been discovered that the loss offrataxin in mitochondria causes the progressive hyperacetylation ofmitochondrial proteins. Accordingly, the present disclosure is directedto methods for screening for diseases and disorders associated with adeficiency in frataxin, as well as to a kit for measuring or detectingprotein acetylation levels. Additionally, the present disclosure isdirected to methods for detecting the progression of a disease ordisorder associated with a deficiency in frataxin in a subject having orsuspected of having a deficiency in frataxin and to methods formonitoring the effectiveness of therapy in a subject having or suspectedof having a disease or disorder associated with a deficiency infrataxin. The present disclosure also is directed to methods ofscreening for therapeutic agents for use in treating diseases anddisorders associated with a deficiency in frataxin.

In one aspect, the present disclosure is directed to a method ofscreening for a deficiency in frataxin in a subject. The method includesdetermining the acetylation status of a mitochondrial protein in asample tissue and determining the acetylation status of a mitochondrialprotein in a normal tissue. An increase in acetyl-lysine in the sampletissue as compared to acetyl-lysine in the normal tissue is indicativeof a deficiency in frataxin.

In another aspect, the present disclosure is directed to a method ofscreening for a deficiency in frataxin in a subject. The method includesdetermining the mitochondrial NADH level in a sample tissue anddetermining the mitochondrial NADH level in a normal tissue. An increasein the NADH level in the sample tissue as compared to the NADH level inthe normal tissue is indicative of a deficiency in frataxin. In someembodiments, the method further includes determining the mitochondrialNAD⁺ level in a sample tissue and/or determining the NAD⁺/NADH ratio inthe sample tissue and determining the mitochondrial NAD⁺ level in anormal tissue and/or determining NAD⁺/NADH ratio in the normal tissue,wherein a decrease in the NAD⁺/NADH ratio in the sample tissue ascompared to the NAD⁺/NADH ratio in the normal tissue is indicative of adeficiency in frataxin.

In another aspect, the present disclosure is directed to a kit formeasuring levels of mitochondrial protein acetylation. The kit includesan arm-acetyl-lysine-specific antibody and an antibody that specificallybinds to a mitochondrial protein. The kit may include reagents forprocessing a sample, for isolating mitochondria, and for detectingprotein acetylation levels (e.g., isolation buffers, colorimetric assaybuffers and reagents, and acetylation detection buffers and reagents).In one embodiment, the kit measures acetyl-lysine residues with, forexample, an acetyl-lysine-specific antibody.

In another aspect, the present disclosure is directed to a method ofdetecting the progression of a disease or a disorder associated with adeficiency in frataxin. The method includes measuring mitochondrialprotein acetylation in an earlier-obtained sample (i.e., a firstchronological sample) and a later-obtained chronological sample (i.e., asecond chronological sample) from a subject having or suspected ofhaving the disease or the disorder associated with a deficiency infrataxin, where an increase in mitochondrial protein acetylation in thesecond chronological sample indicates progression of the disease or thedisorder when compared to the mitochondrial protein acetylation in thefirst chronological sample.

In another aspect, the present disclosure is directed to a method formonitoring the effectiveness of therapy in a subject having or suspectedof having a deficiency in frataxin. The method includes measuringmitochondrial protein acetylation in at least a first chronologicalsample, administering the therapy to the subject, and measuringmitochondrial protein acetylation in at least a second chronologicalsample, where a decrease in the mitochondrial protein acetylation in thesecond chronological sample when compared to the mitochondrial proteinacetylation in the first chronological sample indicates effectiveness oftherapy.

In another aspect, the present disclosure is directed to a method forscreening for agents that can be used for treating a disease or adisorder associated with a deficiency in frataxin. The method includesmeasuring mitochondrial protein acetylation in a subject havinghyperacetylated mitochondrial proteins, administering an agent suspectedof modulating protein acetylation to the subject, measuringmitochondrial protein acetylation after administration of the agent,where the agent is considered to be a candidate for treating the diseaseor the disorder associated with a deficiency in frataxin if themitochondrial protein acetylation is decreased in the sample afteradministration of the agent.

BRIEF DESCRIPTION OF THE DRAWINGS

The disclosure will be better understood, and features, aspects andadvantages other than those set forth above will become apparent whenconsideration is given to the following detailed description thereof.Such detailed description makes reference to the following drawings,wherein:

FIG. 1 is a Western blot (WB) probing for internal acetyl-lysineresidues using total heart homogenates derived from 24 day-old wild-type(WT, n=2, lanes 1 and 2) and 24 day-old NSE-Cre knockout mice (“NSE KO”;n=2, lanes 3 and 4), the corresponding SDS-PAGE gel stained withCoomassie Blue and a Western blot probing for frataxin as discussed inExample 2.

FIG. 2 is a Western blot (WB) probing for internal acetyl-lysineresidues using total heart homogenates prepared from 9 week-old WT (n=2,lanes 1 and 2) and 9 and 11 week-old MCK-Cre mouse models of FA (lanes 3and 4, respectively), the corresponding SDS-PAGE gel stained withCoomassie Blue and a Western blot probing for frataxin as discussed inExample 2.

FIG. 3 is a Western Blot probing for internal acetyl-lysine residues(“Ac-K”) of total heart homogenate (“total”), the cytoplasmic fraction(“cyto”) and the mitochondrial (“mito”) fraction, a Western blot probingfor the mitochondrial marker complex H iron-sulfur subunit, and aWestern blot probing for the cytoplasmic marker GAPDH as discussed inExample 2.

FIG. 4 is a graph showing the average relative integrated densitometryvalues from the acetyl-lysine Western blot M FIG. 1 (mean±SD; *P<0.05)as discussed in Example 2.

FIG. 5 is a graph showing the average relative integrated densitometryvalues from the acetyl-lysine Western blot in FIG. 2 (mean±SD; *P<0.05)as discussed in Example 2.

FIG. 6 is a Western blot (WB) demonstrating the purity of the cardiacmitochondrial preparations using Histone H3 as a nuclear marker, Gapdhas a cytoplasmic marker, VDAC as a mitochondrial outer membrane marker,and Complex H as a mitochondrial inner membrane marker as discussed inExample 3.

FIG. 7 is a Western blot probing for internal acetyl-lysine residues ofisolated mitochondrial protein obtained from hearts from 24 day-oldwild-type (WT, n=2, Lanes 1 and 2) hearts and 24 day-old NSE frataxinknockout hearts (“NSE”; n−2, Lanes 3 and 4), Western blots probing forthe respiratory Complex 1 subunit NDUFA9, the Complex II 30 kDairon-sulfur subunit, SIRT3 and the mitochondrial outer membrane proteinvoltage-dependent anion channel (VDAC) as a loading control as discussedin Example 3.

FIG. 8 is a graph showing the calculated densitometry for SIRT3 relativeto the loading control VDAC shown in FIG. 7 (mean±SD; N.S.: notsignificant) as discussed in Example 3.

FIG. 9 is a Western blot probing for internal acetyl-lysine residues ofmitochondrial protein preparations from WT or NSE-Cre frataxin knockouthearts at day 7 showing an increase in acetylation as early aspost-natal day 7 concomitant with downregulation of the respiratorycomplex II iron-sulfur as discussed in Examples 3 and 4.

FIG. 10 is a Western blot probing for internal acetyl-lysine residues ofmitochondrial protein preparations isolated from 2-3 WT or NSE-Crefrataxin knockout hearts at day 7, day 17, and day 24 in theirpost-natal development, a Western blot probing for frataxin and VDAC asa loading control as discussed in Example 4.

FIG. 11 is a graph assessing the post-natal development of cardiachypertrophy in the NSE KO mice as measured by heart weight divided bybody weight (mean±SD; n=3 measurements at each time point; *P<0.05) asdiscussed in Example 4.

FIG. 12 is a graph showing the determination of nicotinamide adeninedinucleotide (NAD⁺) and reduced NAD⁺ (NADH) levels in WT and frataxindeficient cardiac mitochondrial preparations (n=3-5 biologicalreplicates per condition, mean±SD; **P<0.005) and the figure inset showsthe ratio of NAD⁺ to NADH for WT and NSE-KO heart mitochondria asdiscussed in Example 5.

FIG. 13 is a Western blot showing the carbonyl pulldown of SIRT3 infrataxin-deficient mitochondria as discussed in Example 6.

FIG. 14 is a Western blot showing immunoprecipitation of WT (+/+) andNSE-Cre frataxin-deficient (−/−) cardiac mitochondrial proteins with anacetyl-lysine antibody and subsequent Western blot probing for therespiratory complex I subunit NDUFA9 and Western blots of 2 percent ofthe total mitochondrial lysate input used for the immunoprecipitationsshowing NDUFA9, SIRT3, frataxin, and the acetylation states of bothsamples as discussed in Example 7.

FIG. 15 is a Western blot showing immunoprecipitation of WT (+/+) andNSE-Cre frataxin-deficient (−/−) cardiac mitochondrial proteins with anacetyl-lysine antibody and subsequent Western blot probing foracetyl-CoA synthetase 2. (AceCS2) and Western blots of 2 percent of thetotal mitochondrial lysate input used for the immunoprecipitationsshowing AceCS2, SIRT3, frataxin, and the acetylation states of bothsamples as discussed in Example 7.

FIG. 16 shows Western blots probing for internal acetyl-lysine residuesof mitochondrial protein preparations from WT or NSE-Cre frataxinknockout hearts incubated with recombinant SIRT3 and NAD⁺ showing areduction of the acetyl-lysine signal as discussed in Example 8.

FIG. 17 is a Western blot probing for internal acetyl-lysine residues ofwhole heart lysate derived from a 13 month-old wild-type (WT) animal anda 12 month-old animal harboring a missense mutation in the mitochondrialgenome-encoded protein subunit of the respiratory chain enzymecytochrome c oxidase (COI) and the same membrane used for Westernblotting stained with Ponceau S as discussed in Example 9.

FIG. 18 is a graph showing the calculated integrated densitometry valuesfor acetyl-lysine signal relative to Ponceau S signal from FIG. 17 asdiscussed in Example 9.

FIG. 19 is a Western blot probing for internal acetyl-lysine residues ofwhole heart lysate prepared from a 18 month-old WT animal and a 20month-old animal lacking the non-respiratory chain mitochondrial innermembrane protein adenine nucleotide translocase 1 (ANTI^(−/31)) and thesame membrane used for Western blotting stained with Ponceau S asdiscussed in Example 9.

FIG. 20 is a graph showing the calculated integrated densitometry valuesfor acetyl-lysine signal relative to Ponceau S signal from FIG. 19 asdiscussed in Example 9.

FIG. 21A is a Western blot probed for acetylation of mitochondrialproteins from mice with Friedreich's Ataxia treated with TAT-Frataxin asdiscussed in Example 10.

FIG. 21B is a graph quantifying the amount of acetylation of the uppermost 4 bands shown in FIG. 21A by densitometry as discussed in Example10.

While the disclosure is susceptible to various modifications andalternative forms, specific embodiments thereof have been shown by wayof example in the drawings and are herein described below in detail. Itshould be understood, however, that the description of specificembodiments is not intended to limit the disclosure to cover allmodifications, equivalents and alternatives falling within the spiritand scope of the disclosure as defined by the appended claims.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the disclosure belongs. Although any methods andmaterials similar to or equivalent to those described herein may be usedin the practice or testing of the present disclosure, the preferredmaterials and methods are described below.

In accordance with the present disclosure, biomarkers useful forassessing mitochondrial damage in disease have been discovered.Significantly, it has been discovered that a deficiency in frataxin cancause progressive hyperacetylation of mitochondrial proteins.Accordingly, the present disclosure relates to assessing the acetylationstatus of mitochondrial proteins as biomarkers for assessingmitochondrial damage. Methods for assessing acetylation status of, forexample, mitochondrial proteins to determine oxidative damage tomitochondria in certain diseases have also been discovered.)

Suitable methods for determining the acetylation status of amitochondrial protein may be performed by Western blot analysis,immunoprecipitation, enzyme-linked immunosorbent assay (ELISA) and massspectrometry.

In another aspect, the present disclosure is directed to a method ofscreening for a disease or a disorder associated with a deficiency infrataxin in a subject. The method includes determining the acetylationstatus of a mitochondrial protein, wherein acetyl-lysine in a samplefrom the subject is increased as compared to acetyl-lysine in normaltissue is indicative of a disease or a disorder associated with adeficiency in frataxin. As used herein, “normal tissue” refers to areference sample obtained from a subject that is known to not have adisease or a disorder associated with a deficiency in frataxin. Thephrase “normal tissue” is also intended to refer to a reference valueassigned to a subject or a panel of subjects that are known to not havea disease or a disorder associated with a deficiency in frataxin.

Mitochondria may be isolated by methods for isolating mitochondria fromcells known to those skilled in the art. The method may further includesub-fractionation of the tissue sample. Sub-fractionation of the tissuesample allows for the isolation of mitochondria from other cellular andsub-cellular components of the tissue. Any suitable methods known bythose skilled in the art may he used for tissue sub-fractionation. Forexample, tissue may be homogenized and then subjected to a standarddifferential centrifugation method to initially pellet nuclei andcellular debris followed by centrifugation of the resulting supernatantto pellet mitochondria.

Protein acetylation may be measured by methods for detecting acetylationknown to those skilled in the art. A particularly suitable method fordetecting protein acetylation may be, for example, by detecting lysineacetylation. In one embodiment, levels of acetyl-lysine may be measuredby utilizing an antibody specific for acetyl-lysine residues. Antibodiesthat specifically bind to acetyl-lysine residues are commerciallyavailable (Immunechem, Cell Signaling) or may be made using methodsknown by those skilled in the art. Suitable methods for detecting lysineacetylation status of a mitochondrial protein can be determined, forexample, by Western blot analysis, immunoprecipitation, enzyme-linkedimmunosorbent assay (ELISA) mass spectrometry and combinations of thesemethods.

Suitable subjects may be mammals. Particularly suitable mammals can he,for example, humans and rodents such as, for example, rats and mice.

Mitochondrial proteins that are particularly suitable for determiningacetyl-lysine status include those known by one skilled in the art (seee.g., Hebert, A. S., et al., Mol. Cell. 49:186-199 (2013)).

The diseases and disorders associated with a deficiency in frataxin canbe, for example, Friedereich's Ataxia, Parkinson's Disease, Alzheimer'sDisease, alcoholism, ischemic heart disease, dementia, Huntington'sdisease, Amyotrophic lateral sclerosis, cytochrome c oxidase deficiency,autosomal dominant progressive external ophthalmoplegia (adPEO), andLeber's Hereditary Optic Neuropathy (LHON). Particularly suitablesamples may be a tissue having cells that possess mitochondria and maybe from any part of the body that is affected by the disease.Particularly suitable tissues for obtaining samples can be, for example,liver, heart, white blood cells, and n Lan tissue.

In another aspect, the present disclosure is directed to a method ofdetecting the progression of a disease or a disorder associated with adeficiency in frataxin in a subject. The method includes measuringmitochondrial protein acetylation, especially lysine acetylation, in afirst chronological sample and a second chronological sample from asubject having or suspected of having the disease or the disorderassociated with a deficiency in frataxin. An increase in themitochondrial protein acetylation in the second chronological sampleindicates progression of the disease when compared to mitochondrialprotein acetylation in the first chronological sample. For chronologicalmeasurement to detect the progression of a disease, mitochondrialprotein acetylation can he determined from about one day to about 30days. It is within the skill of those in the art to detect theprogression of a mitochondrial respiratory chain disorder by measuringmitochondrial protein acetylation.

In another aspect, the present disclosure is directed to a method ofmonitoring the effectiveness of a therapy in a subject having orsuspected of having a disease or a disorder associated with a deficiencyin frataxin. The method includes measuring mitochondrial proteinacetylation in at least a first chronological sample, administering thetherapy to the subject and measuring mitochondrial protein acetylationin at least a second chronological sample from the subject. A decreasein the mitochondrial protein acetylation in the second chronologicalsample indicates effectiveness of therapy when compared to themitochondrial protein acetylation in the first chronological sample. Forchronological measurement to monitor the effectiveness of amitochondrial respiratory chain disorder therapy, mitochondrial proteinacetylation can be determined from about one day to about one year. Itis within the skill of those in the art to begin a therapy and monitorthe effectiveness of over the entire course of therapy, as well as for atime following termination of therapy.

In another aspect, the present disclosure is directed to a method forscreening for agents that can be used as therapeutic agents for treatinga disease or a disorder associated with a deficiency in frataxin. Themethod includes measuring mitochondrial protein acetylation in a subjecthaving hyperacetylated mitochondrial proteins, administering an agentsuspected of modulating protein acetylation to the subject, andmeasuring mitochondrial protein acetylation after administration of theagent. The agent is considered to be a candidate for treating thedisease or the disorder associated with a deficiency in frataxin if themitochondrial protein acetylation is decreased in the sample afteradministration of the agent.

In another aspect, the present disclosure is directed to a method ofscreening for a disease or a disorder associated with a deficiency infrataxin in a subject. The method includes determining the mitochondrialNADH level in a sample tissue, wherein an increase in NADH level in thesample tissue as compared to NADH level in normal tissue is indicativeof a mitochondrial respiratory chain disorder.

The method may further include determining mitochondrial NADH level asample tissue, determining NAD⁺/NADH ratio in the sample tissue,determining mitochondrial NAD⁺ level in a normal tissue, and determiningNAD⁺/NADH ratio in the normal tissue, wherein a decrease in theNAD³⁰/NADH ratio in the sample tissue as compared to the NAD³⁰/NADHratio in normal tissue is indicative of a disease or a disorderassociated with a deficiency in frataxin.

NAD⁺ and NADH levels can be done using methods known by those skilled inthe art using commercially available assays (Bioassay Systems). OnceNAD⁺ and NADH levels are determined, the NAD⁺/NADH ratio can becalculated.

In another aspect, the present disclosure is directed to a kit forMeasuring or detecting mitochondrial protein acetylation. The kit mayinclude reagents for processing a sample, for isolating mitochondria,and for detecting protein acetylation levels (e.g, isolation buffers,colorimetric assay buffers and reagents, and acetylation detectionbuffers and reagents). In one embodiment, the kit measures acetyl-lysineresidues with, for example, an anti-acetyl-lysine-specific antibody.Suitable anti-acetyl-lysine antibodies are commercially available(Immunechem; Cell Signaling).

The kit may further use a combination of antibodies such as, forexample, an anti-acetyl-lysine-specific antibody and ananti-mitochondrial protein antibody. Suitable anti-mitochondrial proteinantibodies can be, for example, antibodies that specifically bind toNADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9 (NDUFA9),antibodies that specifically bind to acetyl-CoA synthetase 2 (AceCS2),antibodies that specifically bind to frataxin, antibodies thatspecifically bind to complex II 30 kDa subunit, antibodies thatspecifically bind to complex III Rieske protein, antibodies thatspecifically bind to NAD-dependent deacetylase sirtuin-3 (SIRT3),antibodies that specifically bind to voltage-dependent anion-selectivechannel (VDAC) and antibodies that specifically bind to othermitochondrial proteins known to those skilled in the art. The antibodiesmay be, for example, monoclonal antibodies, polyclonal antibodies andcombinations thereof

Kits may also use solid supports such as, for example, microtiterplates, membranes (e.g., nitrocellulose, polyvinyl chloride, nylon,polyvinylidene fluoride, diazotized paper) and beads (e.g., polystyrenelatex, activated beads, Protein A beads, Protein G beads).

The kit can further include instructions for using the reagents forprocessing a sample, for isolating mitochondria, for measuringmitochondrial protein acetylation, for detecting protein acetylationlevels and combinations thereof. The instructions may be provided in thekit packaging and provided by other media such as, for example, awebsite.

The disclosure will be more fully understood upon consideration of thefollowing non-limiting Examples.

EXAMPLE Example 1 Materials and Methods

Frataxin conditional knockout mouse breeding and genotyping. Mice wereobtained from Helene Puccio and bred as described previously to generatethe NSE and MCK-Cre conditional knockout animals. PCR was used to detectthe presence of a 500 bp product of exon 4 and a 790 bp product of theCre transgene for genotyping. Mice that were genotyped as Frda^(L3/Δ),Cre+ represent the conditional knockout animals.

Mouse tissue preparation. For the isolation of whole heart homogenate,animals were sacrificed by CO₂ asphyxiation and the tissue wasimmediately excised, washed and minced in cold phosphate-bufferedsaline. The tissue was then placed in 1-2 ml of ice-cold complete MPAbuffer containing an EDTA-free protease inhibitor cocktail (Rocheproduct #05056489001), and 10 mM nicotinamide (NAM), 200 nM TrichostatinA, and 5 mM sodium butyrate as deacetylase inhibitors. The tissues werethen homogenized with at least 10 passes of a motor-drivenPotter-Elvehjem homogenizer at high speed. The whole tissue homogenateswere then transferred to 1.5 ml tubes and spun at 17,000×g for 10 min at4° C. in a desktop centrifuge to pellet membranes and tissue debris. Thesupernatant was saved and immediately frozen on dry-ice and stored at−80° C. for later analysis. Frozen, RIPA buffer solubilized ANT1^(−/−)and COI missense mutation cardiac lysates were provided by Dr. DouglasC. Wallace of The Children's Hospital of Philadelphia.

Isolation of Cardiac Mitochondria. Wild-type or frataxin conditional KOmice were sacrificed by CO₂ asphyxiation and their hearts wereimmediately removed and submerged in ice-cold mitochondrial isolationbuffer (MIB) containing 220 mM mannitol, 70 rarVi. sucrose, 30 mMTris-Cl (pH 7.4), 0.5 mM EGTA, and 0.1% BSA. Heart tissue was minced andwashed in MIB to eliminate blood and serum contaminants, then quicklyweighed. Minced heart tissue from 2-3 animals was submerged in 3 ml/gMIB containing an EDTA-free protease inhibitor cocktail (Roche) and 10mM nicotinamide (NAM), 200 nM Trichostatin A, and 5 mM sodium butyrateas deacetylase inhibitors. Heart tissue was homogenized with four passesof a motor-driven Potter-Elvehjem homogenizer with a Teflon pestle atmedium speed on ice. An additional 7 ml/g MIB was added to the hearthomogenate, which was then transferred to a pre-chilled 50 ml conicaltube. A crude mitochondrial pellet was obtained via a standarddifferential centrifugation method. Briefly, nuclei and the heavy cellfraction are removed via centrifugation for 15 minutes at 1000×g at 4°C. The resulting post-nuclear supernatant was subjected tocentrifugation for 15 min at 10,000×g to pellet mitochondria. Thesupernatant was removed as the cytosolic fraction and sedimentedmitochondria were gently resuspended in 1 ml ice-cold MIB containingdeacetylase inhibitors and then diluted with MIB to a total volume of 10ml and centrifuged again for 15 min at 10,000×g. The supernatant wasthen removed and sedimented mitochondria are gently washed in 0.5 M KClto remove endoplasmic reticulum and lysosome contaminants. For Westernblotting, the sedimented mitochondria were solubilized in complete RIPAbuffer containing protease and deacetylase inhibitors (see Mouse tissuepreparation) and frozen until later analysis. For immunoprecipitations,the pelleted mitochondria were resuspended in ice-cold buffer containing50 mM Tris-Cl (pH 7.4), 150 mM NaCl, and an EDTA-free protease inhibitortablet and immediately frozen at −80° C. For NAD⁺ and NADH measurements,sedimented mitochondria were immediately resuspended in 180 μl ofice-cold 1 mM Tris-Cl pH 7.4, 150 mM NaCl and extracted appropriately(see Mitochondrial NAD⁺ and NADH measurements).

Antibodies and Western Blotting. Antibodies used includedanti-acetyl-lysine (Immunechem anti-acetyl-lysine (Cell Signaling)anti-complex II 30 kDa subunit, anti-NDUFA9, anti-complex III Rieskeprotein (Mitosciences), anti-SIRT3 (provided by E. Verdin, GladstoneInstitute, UCSF), anti-frataxin (provided by G. Isaya, Mayo Clinic),anti-SIRT3 D22A3, anti-VDAC D73D12, and anti-histone H3 (CellSignaling), anti-GAPDH and anti-tubulin (Sigma), and anti-AceCS2 (ACSS1)(Abeam). Following transfer of the SDS-PAGE gel proteins tonitrocellulose membranes, the membrane was blocked for 45 min inPBS-0.05% Tween-20 supplemented with 5% non-fat dry milk. Signals werevisualized with SuperSignal West chemiluminescent substrate(Thermo-Pierce). Western blot signal intensities were determined bydensity quantification using ImageJ software where appropriate. Proteinconcentrations were determined using the BCA method (Pierce).

Immunoprecipitation of acetyWysine proteins. For immunoprecipitation ofacetyl-lysine proteins and detection of NDUFA9 and AceCS2 acetylation,500 μg to 1 mg of cardiac mitochondria was solubilized for 30 min on icein 1% lauryl maltoside (Mitosciences) and centrifuged at 16,000×g for 10min at 4° C. to pellet debris and lipid membrane components.Anti-acetyl-lysine antibody (Cell Signaling) was loaded with thesolubilized mitochondrial supernatant at a ratio of 1:25 w/w andincubated overnight at 4° C. with gentle agitation. Following incubationwith the anti-acetyl-lysine antibody, the sample was incubated at 4° C.with gentle agitation for an additional 2 hours with 7.5 μl Protein Aagarose (Invitrogen) plus 7.5 μl Protein G agarose (Roche). Agarose wascollected via a 1 min centrifugation at 400×g at 4° C. with a desktopcentrifuge and the unbound mitochondrial supernatant was carefullyremoved from above the agarose beads. The beads were washed 3 times for5 min. in 500 μl IP buffer containing 50 mM Tris-Cl (pH 7.4), 150 mMNaCl, 1 mM EGTA, and 0.5% NP-40 at 4° C. and then incubated in 30 μl SDSsample loading buffer and boiled at 95° C. for 5 min. Theimmunoprecipitated sample was then resolved on a 12% SDS-PAGE andprocessed for Western blotting.

Mitochondrial NAD⁺ and NADH measurements. NAM was excluded from MIB usedto isolate mitochondria for NAD⁺ measurements as the enzyme nicotinamidephosphoribosyltransferase (Nampt) catalyzes the formation of NMN, anNAD⁺ precursor, from NAM. NAD(H) measurements were performed with acycling assay according to the manufacturer's instructions (BioassaySystems). NAD⁺ was extracted in the mitochondrial sample via addition ofstrong acid and then heated at 60° C. to eliminate the presence of thereduced nucleotide (NADH). NADH was extracted in mitochondrial samplehomogenate via addition of strong base and then heated at 60° C. toeliminate the presence of the oxidized nucleotide (NAD⁺). Theextractions were neutralized with the opposite extraction buffer and thedehydrogenase cycling assay was performed in which, in the presence oflactate, lactate dehydrogenase, diaphorase and MTT formazan reagent, theconcentration of NAD⁺ or NADH present in the sample is proportional tothe amount of reduced TvITF formation which was colorimetricallydetected with a plate reader at 562 nm. Sample concentrations weredetermined via an NAD⁺ standard curve. The coenzyme concentration wasnormalized to the protein concentration of the sample.

In vitro SIRT3 deacetylation assay 200 μl of 2 μg/μl Frataxin cardiacmitochondria frozen in 50 mM Tris-Cl (pH 7.4). 1.50 mM NaCl was thawedon ice and solubilized in 1% lauryl maltoside for 30 min on ice andcentrifuged at 16,000×g for 10 min at 4° C. to pellet debris and lipidmembrane components. 15 μl of the soluble mitochondrial supernatant wasmixed 1:1 with 15 μl of 2× deacetylase buffer (50 mM Tris-Cl pH 8.0, 150mM NaCl, 2 mM MgCl₂, with or without 20 mM NAD⁺). 3 μg of recombinantGST-tagged SIRT3 corresponding to amino acids 101-399 of human SIRT3(Sigma) in 40 mM Tris-Cl pH 8.0, 240 mM NaCl, and 20% glycerol was addedto 30 μl (30 μg,) of the 1:1 mitochondrial protein:2× deacetylase buffermix in a 1.5 ml tube. An appropriate amount of buffer containing only 40mM Tris-Cl, 240 mM NaCl, and 20% glycerol was added to incubationslacking SIRT3 to equalize the incubation volumes and conditions. Thereactions were incubated for 4 hours at 37° C. at 400 rpm in anEppendorf Thermomixer and briefly centrifuged every 30 min of incubationtime to minimize reaction condensation. Following the incubation, thereactions were stopped upon addition of SDS loading buffer, boiled andprocessed for Western blotting as described previously.

Detection of 4-HNE modified SIRT3. For the detection of 4-HNE modifiedSIRT3, cardiac mitochondrial proteins were solubilized as describedearlier and 4-HNE modified proteins were derivatized to a biotin groupvia a 2 hour incubation in 5 mM EZ-Link hydrazide biotin (ThermoScientific) in the dark. The samples were dialyzed 1:10000 in PBSovernight to remove excess biotin hydrazide. 100 μg of protein samplewas then incubated with 50 μl of NeutrAvidin Agarose Resin (ThermoScientific) at 4° C. with gentle rocking overnight. Beads were washed 5times for 5 mM with 500 μl PBS-0.2% Tween 20, then boiled at 95° C. in35 μl SDS loading buffer and processed for Western blotting as describedpreviously.

Statistical Analysis. Statistical significance was determined using atwo sample t-test assuming unequal variances. Any P value less than 0.05was judged to be significant.

Example 2

In this Example, NSE- and MCK-Cre conditional mouse models forFriedreich's Ataxia (FRDA) were analyzed for protein acetyl-lysinemodifications.

Specifically, whole heart lysates from wild-type (WT), NSE, and MCKconditional mouse models of FRDA were analyzed by Western blot analysisto assay protein acetyl-lysine modifications. As shown in FIGS. 1 and 2,heart lysates from both the NSE (NSE KO) and MCK (MCK KO) mouse modelsof FRDA exhibited marked increases in acetyl-lysine modifications ascompared to age-matched control hearts. As demonstrated in FIG. 3,hyperacetylation was localized to cardiac mitochondria. As shown inFIGS. 4 and 5, the increases in acetyl-lysine modifications as comparedto age-matched control hearts were statistically significant. Thedifferences were most dramatic in proteins with an estimated molecularweight between approximately 30 and 75 kDa (FIG. 1).

Example 3

In this Example, sub-fractionation of heart samples was performed todetermine the sub-cellular distribution of hyperacetylated proteins.

Analysis of the purity of these mitochondrial preparations usingantibodies to Historic H3 (a cytoplasmic marker), Gapdh (a cytoplasmicmarker), VDAC (a mitochondrial marker) and Complex II (a mitochondrialmarker) showed that nuclear and cytosolic proteins were excluded andthey were highly enriched for markers of both the outer and innermitochondrial membranes (FIG. 6). Using day 24 wild-type (WT, n=2)control and NSE-Cre frataxin-deficient cardiac mitochondrialpreparations (n=2), Western blot analysis was performed foracetyl-lysine modifications.

Control cardiac mitochondria exhibited several acetylated proteinsdetectable by Western blot. However, frataxin-deficient cardiacmitochondria displayed marked hyperacetylation of numerous proteins(FIGS. 3 and 7). This was accompanied by a characteristic downregulationof respiratory complex I and II (succinate dehydrogenase) that waspresent as early as 7 days post-natal (FIG. 9). Levels of the dominantmitochondria-localized NAD⁺-dependent deacetylase SIRT3 displayed amild, though insignificant, increase in frataxin-deficient mitochondrialpreparations (FIGS. 7 and 8). These results indicated that thehyperacetylation observed at the level of whole cardiac lysate waspredominantly localized to mitochondria.

Example 4

In this Example, the developmental profile of mitochondrial proteinacetylation was determined.

Specifically, the NSE-Cre mouse models of FRDA begin to develop cardiachypertrophy in the second week of life. Thus, cardiac mitochondria fromWT control and NSE-Cre mice at post-natal days 7, 17, and 24 wereprepared and analyzed by Western blot analysis.

As shown in FIGS. 9 and 10, at post-natal day 7, NSE-Cre cardiacmitochondrial proteins exhibited only a mildly increased acetylationstate as compared to their wild-type (WT) counterparts. However, atpost-natal day 17, NSE-Cre cardiac mitochondria displayed increasedacetylation of cardiac mitochondrial proteins as compared to their WTcounterparts, which became more dramatic by post-natal day 24. Theprogressive increase in cardiac mitochondrial protein acetylation overthis time frame corresponded with the development of cardiac hypertrophyin the NSE-Cre models (FIG. 11).

Example 5

In this Example, the redox states of WT and frataxin deficient cardiacmitochondria preparations were determined.

Individual measurements were performed on a pooled sample of at least300 μg of fresh cardiac mitochondria derived from 2-3 hearts andnormalized to total mitochondrial protein input. As shown in FIG. 12, WTheart mitochondria displayed robust NAD⁺ levels and over 100-fold lessNADH by comparison, which is consistent with highly oxidative cardiaccatabolism and a continuous demand for carbon fuels, Frataxin deficientmitochondria displayed a mild, though insignificant, increase in NAD⁺levels when compared to WT. In contrast, frataxin deficientmitochondrial NADH levels were, on average, over 95-fold greater than inWT mitochondria (P<0.005) resulting in a corresponding NAD⁺/NADH ratiothat was 85-fold less than in WT animals (FIG. 12 inset). The observedaccumulation of NADH and consequent shift in mitochondrial redox stateis consistent with the impairments of mitochondrial respiration found inboth human FRDA patients, and the NSE and MCK animal models.

Example 6

In this Example, differential modification of SIRT3 by 4-HNE in frataxindeficient cardiac mitochondria was determined.

Specifically, all reactive aldehyde containing mitochondrial proteinsfollowing their derivatization to biotin hydrazide were pulled down, andthen analyzed by Western blot for SIRT3. As shown in FIG. 13, SIRT3 inWT heart mitochondria exhibited a small amount of 4-HNE modification. Incontrast, SIRT3 in frataxin-deficient mitochondria exhibited a markedincrease in 4-HNE modification suggesting that SIRT3 may be directlyinhibited via carbonyl group adduction in the setting of frataxindeficiency.

Example 7

In this Example, the acetylation states of known targets ofSIRT3-mediated deacetylation was determined.

Mitochondrial acetyl-lysine proteins were immunoprecipitated andanalyzed by Western blot for NDUFA9. Consistent with previous findings,a minimal amount of NDUFA9 acetyl-lysine signal in the WT (+/+)mitochondrial immunoprecipitate was observed. However, thefrataxin-deficient (−/−) mitochondrial immunoprecipitate displayed agreater acetylated NDUFA9 signal despite downregulation of NDUFA9 asseen in the input (FIG. 14). The acetylation state of acetyl-CoAsynthetase 2 (AceCS2) was also analyzed. Similarly, this analysisrevealed a greater amount of acetylated AceCS2 in the frataxin-deficient(−/−) condition as compared to the WT (+/+) (FIG. 15). The increasedacedation states of two known targets of SIRT3-mediated deacetylationindirectly demonstrated that SIRT3 is inhibited in frataxin-deficientcardiac mitochondria. Importantly, increases in the acetylation statesof NDUFA9 and AceCS2 were linked to a decrease in the activity ofrespiratory complex I and the synthesis of activated acetate,respectively.

Example 8

In this Example, the rescue of inhibited endogenous SIRT3 by theaddition of SIRT3 and NAD⁺ in vitro was determined.

Specifically, solubilized WT and frataxin-deficient cardiacmitochondrial proteins were incubated with glutathione S-transferasetagged, processed human recombinant SIRT3 (GST-hSIRT3) in the presenceor absence of NAD⁺ to assay for changes in acetylation states.Incubating solubilized WT or frataxin cardiac mitochondrial homogenateswith NAD⁺ alone caused no change in acetylation signal. In contrast,incubating frataxin deficient mitochondrial protein with 3 μg ofGST-hSIRT3 and NAD⁺ resulted in a marked reduction of acetyl-lysinesignal from nearly every protein band, while completely eliminating theacetyl-lysine signal of multiple bands (FIG. 16). Furthermore, theobserved reduction in acetylation signal was abolished upon withdrawalof NAD⁺ from the incubation buffer, demonstrating that thehyperacetylated protein lysine residues in frataxin-deficient cardiacmitochondria were specifically sensitive to NAD⁺-dependentSIRT3-mediated deacetylation and that the observed hyperacetylation wasnot caused by a general increase in non-specific lysine acetylation.Taken together, these data strongly suggest that hyperacetylation infrataxin deficient mitochondria was due to both redox-state andlipid-peroxidation-mediated inhibition of endogenous SIRT3.

Example 9

In this Example, alterations in cardiac acetylation profiles of othermitochondrial disorders caused by respiratory chain defects weredetermined

Whole cardiac lysates were obtained from the cytochrome oxidase I (COI)mouse model, which harbors a mtDNA mutation causing a defect ofrespiratory complex IV, and the adenine nucleotide translocase knockout(ANT1^(−/−)) mouse model, which models a non-respiratory chain defectwith deficiency of ATP translocation. These mice model the humanmitochondrial disorders, autosomal dominant progressive externalophthalmoplegia (adPEO) and Leber's Hereditary Optic Neuropathy (LHON),respectively, and both models develop cardiomyopathy.

Western blot analysis of cardiac acetylation revealed mild to moderateincreases in acetylation in 12 month old COI hearts as compared to 13month-old control hearts (FIGS. 17 and 18). Interestingly, 20 month oldANT1^(−/−) hearts exhibited a small decrease in acetyl-lysine profileswhen compared to 18 month old control hearts (FIGS. 19 and 20). Takentogether with data from the frataxin deficient mice, these resultssuggest that increases in protein acetylation may be a common feature ofrespiratory chain malfunction in the mammalian heart.

Example 10

In this Example, mitochondrial acetylation status in frataxin-deficientmice treated with TAT-Frataxin fusion protein was determined.

The TAT-Frataxin fusion protein was prepared as described in Vyas et al.(Hum. Mol. Genet. 2012, 21(6):1230-1247). NSE-Cre Frataxin knockout mice(FXN^(−/−)) were treated with TAT-Frataxin for 2 weeks(FXN^(−/−)+TAT-FXN; FIG. 21A lanes 3 and 4) or phosphate buffered saline(FXN^(−/−); FIG. 21A lanes 1 and 2). Specifically, mice were dosedaccording to body weight with a total volume of approximately 20 μl/gramof weight administered intraperitoneally. Whole heart lysate from 4 weekold NSE-Cre FXN knockout mice was quantified and equal loading of eachlane was verified by Coomassie staining. Western blots were probed withanti-acetyl lysine antibody and pixel intensity was quantified for thetop 4 bands identified in lanes 1-4 of FIG. 21A.

As illustrated in FIG. 21B, treatment of mice TAT-Frataxin decreasedprotein acetylation in NSE-Cre FXN knockout mice (NSE KO+TAT-FXN).Therefore, mitochondrial protein acetylation status can be used as abiomarker to monitor treatment in a mouse knockout model of Friedreich'sAtaxia, which is associated with a deficiency in frataxin.

The Examples described above demonstrate that a deficiency in frataxincan cause progressive hyperacetylation of mitochondrial proteins. Morespecifically, hyperacetylation of mitochondrial proteins may be due tothe inhibition of a SIRT3 deacetylase. SIRT3 may respond to the flux ofmitochondrial. NAD⁺ and NADH, which is determined, in large part, by thecapacity of the respiratory chain to oxidize NADH. This capacity isseverely decreased in FRDA, as well as in other mitochondrial defectssuch as cytochrome c oxidase (complex IV) deficiency, causing anaccumulation of NADH and, consequently, a redox state of perceivednutrient excess. It has now been discovered that hyperacetylation ofnumerous mitochondrial proteins correlates with the inhibition of theNAD⁺-dependent SIRT3 deacetylase. This inhibition is caused by an85-fold decrease in mitochondrial NAD⁺/NADH ratio and direct carbonylgroup modification of a NAD-dependent deacetylase. It has also beendiscovered that the inhibition may be rescued by the administration of aNAD-dependent deacetylase and NAD⁺. It has further been discovered thatprotein hyperacetylation provides a specific biomarker of a deficiencyin frataxin. Thus, loss of frataxin causes hyperacetylation of proteinsbecause the respiratory chain is defective and the Krebs cycle.

In view of the above, it will be seen that the several advantages of thedisclosure are achieved and other advantageous results attained. Asvarious changes could be made in the above devices and methods withoutdeparting from the scope of the disclosure, it is intended that allmatter contained in the above description and shown in the accompanyingdrawings shall be interpreted as illustrative and not in a limitingsense.

Then introducing elements of the present disclosure or the variousversions, embodiment(s) or aspects thereof, the articles “a”, “an”,“the” and “said” are intended to mean that there are one or more of theelements. The terms “comprising”, “including” and “having” are intendedto be inclusive and mean that there may be additional elements otherthan the listed elements.

What is claimed is:
 1. A method of screening for a disease or a disorderassociated with a deficiency in frataxin in a subject, the methodcomprising: determining the acetylation status of a mitochondrialprotein in a sample tissue; and determining the acetylation status of amitochondrial protein in a normal tissue, wherein an increase inacetyl-lysine status of the mitochondrial protein in the sample tissueas compared to acetyl-lysine status of the protein in the normal tissueis indicative of the disease or the disorder associated with thedeficiency in frataxin.
 2. The method of claim 1, wherein the sampletissue and the normal tissue is selected from the group consisting ofliver, heart, white blood cells, and neural tissue.
 3. A method ofscreening for a disease or a disorder associated with a deficiency infrataxin in a subject, the method comprising: determining themitochondrial NADH level in a sample tissue; and determining themitochondrial NADH level in a normal tissue, wherein an increase in theNADH level in the sample tissue as compared to the NADH level in thenormal tissue is indicative of the disease or the disorder associatedwith the deficiency in frataxin.
 4. The method of claim 3, furthercomprising determining the NAD⁺ level in the sample tissue anddetermining the NAD⁺ level in the normal tissue.
 5. The method of claim4, further comprising determining the NAD⁺/NADH ratio in the sampletissue and determining the NAD⁺/NADH ratio in the normal tissue, whereina decrease in the NAD⁺/NADH ratio in the sample tissue as compared tothe NAD⁺/NADH ratio in normal tissue is indicative of a disease or adisorder associated with a deficiency in frataxin.
 6. A kit formeasuring levels of mitochondrial protein acetylation comprising ananti-acetyl-lysine-specific antibody and an antibody that specificallybinds to a mitochondrial protein.
 7. The kit of claim 6, wherein theantibody that specifically binds to a mitochondrial protein is selectedfrom the group consisting of an antibody that specifically binds to NADHdehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9 (NDUFA9), anantibody that specifically binds to acetyl-CoA synthetase 2 (AceCS2), anantibody that specifically binds to frataxin, an antibody thatspecifically binds to complex II 30 kDa subunit, an antibody thatspecifically binds to complex HI Rieske protein, an antibody thatspecifically binds to NAD-dependent deacetylase sirtuin-3 (SIRT3), anantibody that specifically binds to voltage-dependent anion-selectivechannel (VDAC), and combinations thereof
 8. The kit of claim 6 furthercomprising at least one of a reagent for processing a sample, a reagentfor isolating mitochondria, a reagent for measuring mitochondrialprotein acetylation, and a reagent for measuring mitochondrial proteinacetylation.
 9. A method of detecting progression of a disease or adisorder associated with a deficiency in frataxin in a subject having orsuspected of having the disease or the disorder associated with thedeficiency in frataxin, the method comprising: measuring mitochondrialprotein acetylation in a first chronological sample from the subject;and measuring mitochondrial protein acetylation in a secondchronological sample from the subject, wherein an increase inmitochondrial protein acetylation in the second chronological sample ascompared to the mitochondrial protein acetylation in the firstchronological sample indicates progression of the disease or thedisorder.
 10. The method of claim 9, wherein the disease or the disorderassociated with the deficiency in frataxin is selected from the groupconsisting of Friedreich's Ataxia, Parkinson's Disease, Alzheimer'sDisease, alcoholism, ischemic heart disease, dementia, Huntington'sdisease, Amyotrophic lateral sclerosis, cytochrome c oxidase deficiency,autosomal dominant progressive external ophthalmoplegia, and Leber'sHereditary Optic Neuropathy.
 11. The method of claim 9, furthercomprising. prior to the measuring step, steps of: collecting the samplefrom the subject; and isolating mitochondria front the sample.
 12. Themethod of claim 9, wherein the subject is a human.
 13. The method ofclaim 9, wherein mitochondrial protein acetylation is measured utilizingWestern blotting with an antibody that detects acetyl-lysine residues.14. A method of monitoring effectiveness of a therapy in a subjecthaving or suspected of having a disease or a disorder associated with adeficiency in frataxin, the method comprising: measuring mitochondrialprotein acetylation in at least a first chronological sample;administering the therapy; measuring mitochondrial protein acetylationin at least a second chronological sample from the subject, analyzingthe mitochondrial protein acetylation in at least the firstchronological sample and the mitochondrial protein acetylation in atleast the second chronological sample, wherein a decrease inmitochondrial protein acetylation in the second chronological sample ascompared to mitochondrial protein acetylation in a first chronologicalsample indicates effectiveness of the therapy.
 15. The method of claim14, wherein the disease or the disorder associated with the deficiencyin frataxin is selected front the group consisting of Friedreich'sAtaxia, Parkinson's Disease, Alzheimer's Disease, alcoholism, ischemicheart disease, dementia, Huntington's disease, Amyotrophic lateralsclerosis, cytochrome c oxidase deficiency, autosomal dominantprogressive external ophthalmoplegia, and Leber's Hereditary OpticNeuropathy.
 16. A method of screening for agents useful for treating adisease or a disorder associated with a deficiency in frataxin, themethod comprising: measuring mitochondrial protein acetylation in asubject having hyperacetylated mitochondrial proteins; administering anagent suspected of modulating protein acetylation to the subject; andmeasuring mitochondrial protein acetylation after administration of theagent, wherein the agent is considered to be a candidate for treatingthe disease or the disorder associated with the deficiency in frataxinif the mitochondrial protein acetylation is decreased in the sampleafter administration of the agent.